After entering tissues, pathogenic bacteria are ingested and killed by mononuclear phagocytes and by granulocytes that constitutively express the pore-forming protein Perforin-2. We show that genetic deficiency or siRNA knock down of Perforin-2 disables killing of the pathogen by professional phagocytes resulting in intracellular replication. Pathogenic bacteria also invade and are endocytosed by epithelial cells and other tissue forming cells. We show that keratinocytes express Perforin-2 constitutively and that all other epithelial and tissue forming cells analyzed are induced to express Perforin-2 by interferons or bacterial invasion. As in phagocytes, siRNA knock down or genetic deficiency of Perforin-2 in tissue forming cells enables intracellular replication of the pathogen which is kille in the presence of Perforin-2. Further analysis reveals that reactive oxygen and nitrogen species and lysosomal hydrolases enhance the bactericidal activity of Perforin-2 but are unable to clear intracellular pathogens without Perforin-2. Genetic deficiency of Perforin-2 in mice causes lethal susceptibility to infection with low doses of Salmonella, Staphylococcus and other pathogens that are cleared in Perforin-2 sufficient animals. Perforin-2 is a MACPF domain containing, integral membrane protein; its activation and killing mechanisms is highly complex. We show that Perforin-2 is localized in membrane vesicles that are distributed throughout the cytosol in uninfected cells. Bacterial infection causes rapid accumulation of Perforin-2 in the phagosomal or endosomal membrane enclosing the bacterium. Re-isolation of bacteria from infected cells and electron microscopic analysis reveals clusters of 100 ? wide pores on bacterial cell walls suggesting that the lethal hit is mediated by polymerization and pore-formation by Perforin-2. Pathogenic bacteria have mechanisms to evade or subvert Perforin-2 in order to survive inside cells. In this application we will study the molecular mechanisms of Perforin-2 activation and killing, which is requisite for development of treatments to enhance Perforin-2 mediated killing that may overcome antibiotic resistance.